U.S. patent number 5,627,160 [Application Number 08/098,650] was granted by the patent office on 1997-05-06 for l-2',3'-dideoxy nucleoside analogs as anti-hepatitis b (hbv) and anti-hiv agents.
This patent grant is currently assigned to Yale University. Invention is credited to Yung-Chi Cheng, Tai-Shun Lin.
United States Patent |
5,627,160 |
Lin , et al. |
May 6, 1997 |
L-2',3'-dideoxy nucleoside analogs as anti-hepatitis B (HBV) and
anti-HIV agents
Abstract
The present invention relates to the surprising discovery that
certain dideoxynucleoside analogs which contain a dideoxy
ribofuranosyl moiety having an L-configuration (as opposed to the
naturally occurring D-configuration) exhibit unexpected activity
against Hepatitis B virus (HBV). In particular, the compounds
according to the present invention show potent inhibition of the
replication of the virus in combination with very low toxicity to
the host cells (i.e., animal or human tissue). Compounds according
to the present invention exhibit primary utility as agents for
inhibiting the growth or replication of HBV, HIV and other
retroviruses, most preferably HBV. The compound
1-(2,3-dideoxy-beta-L-ribofuranosyl)-5-fluorocytosine is shown to
be a potent anti-HIV agent with low toxicity to host cells.
Inventors: |
Lin; Tai-Shun (North Haven,
CT), Cheng; Yung-Chi (Woodbridge, CT) |
Assignee: |
Yale University (New Haven,
CT)
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Family
ID: |
26747712 |
Appl.
No.: |
08/098,650 |
Filed: |
July 28, 1993 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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67299 |
May 25, 1993 |
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Current U.S.
Class: |
514/49; 514/885;
536/28.1; 536/28.5; 536/28.52; 536/28.2 |
Current CPC
Class: |
C07D
409/04 (20130101); A61P 31/20 (20180101); A61P
1/16 (20180101); C07H 19/06 (20130101); A61P
31/12 (20180101); A61P 31/18 (20180101); C07H
19/16 (20130101); Y10S 514/885 (20130101) |
Current International
Class: |
C07D
409/00 (20060101); C07D 409/04 (20060101); C07H
19/16 (20060101); C07H 19/00 (20060101); C07H
19/06 (20060101); A61K 031/70 (); C07H
019/06 () |
Field of
Search: |
;536/28.1,28.2,28.5,28.52 ;514/45,49,50,885 |
References Cited
[Referenced By]
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WO |
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|
Primary Examiner: Wilson; James O.
Attorney, Agent or Firm: Coleman; Henry D. Sudol; R.
Neil
Government Interests
This work is supported by National Institutes of Health Grants
AI29430 and CA44358. The United States Government retains certain
rights in the invention.
Parent Case Text
RELATED APPLICATIONS
This application is a continuation-in-part application of U.S.
patent application Ser. No. 08/067,299, entitled "Novel
L-2',3'-Dideoxy Nuceloside Analogs as Anti-Hepatitis (HBV) Agents",
filed May 25, 1993.
Claims
We claim:
1. A method for treating a patient infected with human
immunodeficiency virus comprising administering to said patient an
amount of
1-(2,3)-didehydro-dideoxy-beta-L-ribofuranosyl)-5-fluorocytosine
effective for inhibiting the growth or replication of said
virus.
2. The method according to claim 1 wherein said compound is
administered in combination with a pharmaceutically acceptable
additive or excipient.
3. The method according to claim 1 wherein said compound is
administered in oral dosage form.
4. The method according to claim 1 wherein said compound is
administered in parenteral dosage form.
Description
FIELD OF THE INVENTION
This invention relates to dideoxy nucleoside analogs. These
compounds exhibit significant activity against retroviruses, and in
particular, Hepatitis B virus. This invention also relates to
pharmaceutical compositions containing these compounds and to
methods of inhibiting the growth or replication of Hepatitis B
virus as well as treating Hepatitis B viral infections in animals
and in particular, humans.
BACKGROUND OF THE INVENTION
Hepatitis B virus (HBV) infection is a major health problem
throughout the world. HBV is a causative agent of both an acute and
chronic form of hepatitis. It is estimated that more than 200
million people worldwide are chronic carriers of HBV.
HBV belongs to the family Hepadnaviridae, which includes a number
of related viruses which primarily infect small rodents. All
members of the hepadnavirus family have a number of characteristics
in common such as morphological appearance, antigenic makeup and
DNA size and structure. Pathological findings following infection
with the members of this family are quite similar. Studies show
that the replication and spread of the viruses of this family are
dependent upon the reverse transcriptase of an RNA
intermediate.
HBV itself is a double-stranded DNA virus. Its DNA polymerase
catalyzes both DNA-dependent and RNA-dependent RNA synthesis. The
life cycle of HBV involves the enzyme reverse transcriptase in its
DNA replication. There is presently no effective drug for the
treatment of an HBV infection.
The best defense against Hepatitis B viral infection is
vaccination. However, even with the advent of immunization
programs, the disease remains a severe worldwide problem. Although
acute Hepatitis B viral infections are generally self-limiting, in
many instances the disease can progress to the chronic state. A
Hepatitis B viral infection also creates a risk to fulminant
hepatitis. In addition, Hepatitis B viral infections are closely
associated with hepatocellular carcinoma.
Present therapy for the treatment of chronic Hepatitis B viral
infections includes the administration of interferon alpha, and
various nucleoside analogs such as adenine arabinoside or its
monophosphate (ara-AMP). These therapeutic approaches have met with
limited success. The use of AZT, acyclovir and foscarnet (in the
case of fulminant hepatitis) to treat hepatitis has also been tried
with little, if any, success.
Several 2',3'-dideoxynucleoside analogs have been reported in the
literature to exhibit potent activity against Hepatitis B virus in
culture. In particular, the nucleoside analogs (+) and
(-)-2',3'-Dideoxy-3'-thiacytidine ((.+-.) SddC) have shown to be
potent inhibitors of Hepatitis B virus and the (-)isomer was
particularly interesting in that it exhibited relatively low
toxicity along with its potent activity. The 5-fluoro analog
((.+-.)5-FSddC) was also shown to exhibit potent activity. (Chang,
et al., Jour. Biol. Chem., 267, 222414, 1992 and Chang, et al.,
Jour. Biol. Chem., 267, 13938, 1992).
Another viral disease which recently has been studied greatly and
treated with only limited success is AIDS. AIDS is a generally
fatal disease caused by a human pathogenic retrovirus known as
human T-lymphotropic virus type III (HTLV III),
lymphadenopathy-associated virus (LAV) or human immunodeficiency
virus (HIV).
In comparison with the other T-lymphotropic retroviruses HTLV I and
II, HTLV III (HIV) and lymphoadenopathy viruses are nontransforming
cytopathic viruses without immortalizing activity. The viral
replication process is believed to be an important event in the
progress of AIDS. It is further believed that the enzyme reverse
transcriptase plays an essential role in the elaboration and life
cycle of HIV and consequently, the progress of the disease. It is
therefore believed that this enzyme may be a particularly
appropriate target for the development of potential drugs against
AIDS because of the absence of such an enzyme in the uninfected
host cell. Recently, investigators have studied a number of
anti-viral agents as potential anti-AIDS agents, including
ribavirin and suramin, among others.
A number of nucleosides have played important roles in the
treatment of HIV infections. 3'-azido-3'deoxythymidine (AZT) is a
prime example, although recent reports raise some doubts about its
effectiveness. A number of 2',3'-dideoxynucleoside analogs also
have exhibited significant activity against human immunodeficiency
virus (HIV), including 3'-deoxy-2',3'-didehydrothymidine (D4T),
carbocyclic analog of 2',3'-dideoxy-2',3'-didehydroguanosine
(Carbovir), 2',3'-dideoxycytidine (DDC),
3'-azido-2',3'-dideoxyguanosine (AZG), 2',3'-dideoxyinosine (DDI),
2',3'-dideoxy-2',3'-didehydrocytidine (D4C),
3'-fluoro-2',3'-dideoxyadenosine, 3'-fluoro-3'-deoxythymidine and
3'-azido-2',3'-dideoxyuridine. See, Larder, et al., Antimicrob.
Agents Chemother., 34, 436 (1990). Certain of these analogs,
including ddC, are currently used as anti-HIV agents. Among the
dideoxynucleosides, ddC has been shown to be among the most potent
inhibitors of HIV.
Although research has concentrated on discovering an effective
treatment protocol against HBV and HIV and certain potent anti-HBV
and anti-HIV nucleoside analogs have been synthesized and
characterized, an ideal drug has not been found.
The major problem in optimizing a treatment protocol against
retroviral infections, including HBV and HIV, is to provide
acceptable anti-viral activity while minimizing the toxicity to the
host cell as well as the anti-mitochondrial DNA effects that many
present anti-viral nucleosides exhibit.
The present invention relates to synthetic nucleosides which
exhibit potent anti-viral activity (in particular, anti-HBV and
anti-HIV activity) with significantly reduced toxicity to the host
cell. In contrast to the prior art compounds, the analogs of the
present invention represent a viable medicinal therapeutic approach
to HBV infections and an improved approach to the inhibition of HIV
and the treatment of AIDS.
OBJECT(S) OF THE INVENTION
It is an object of the present invention to provide nucleoside
compounds which may be used to inhibit the growth or replication of
HIV or other retroviruses.
It is another object of the present invention to provide compounds
which exhibit significant inhibitory activity against HBV or HIV
while minimizing toxicity to the host.
It is a further object of the present invention to provide a method
for inhibiting the growth or replication of HIV.
It is yet another object of the present invention to provide a
therapeutic method for treating HIV infections in humans.
One or more of these and other objects of the present invention may
be readily gleaned from a detailed reading of the description of
the present invention which follows.
BRIEF DESCRIPTION OF THE INVENTION
The present invention relates to the surprising discovery that
certain dideoxynucleoside analogs which contain a dideoxy
ribofuranosyl moiety having an L-configuration (as opposed to the
naturally occurring D-configuration) exhibit unexpected activity
against Hepatitis B virus (HBV). In particular, the compounds
according to the present invention show potent inhibition of the
replication of the virus in combination with very low toxicity to
the host cells (i.e., animal or human tissue). This is an
unexpected result.
Compounds according to the present invention exhibit primary
utility as agents for inhibiting the growth or replication of HBV,
HIV and other retroviruses, most preferably HBV. Certain of these
agents also may be useful for inhibiting the growth or replication
of other viruses or for treating other viral infections, certain
types of fungal infections, microbial infections and/or related
disease states. In addition, certain of these agents may be useful
as intermediates for producing or synthesizing related chemical
species.
Compounds of the present invention find particular use in combating
viral infections which afflict animals, and in particular, humans
suffering from hepatitis B viral infections. Compounds according to
the present invention offer great potential as therapeutic agents
against a disease state (chronic HBV infection) for which there
presently are few real therapeutic options. The compounds according
to the present invention may be used alone or in combination with
agents or other therapeutic treatments.
The compounds of the present invention are dideoxynucleoside
analogs which contain a dideoxyribofuranosyl moiety having an
L-configuration (in contrast to the natural D-configuration of the
sugar moiety). Compounds according to the present invention are
disclosed which contain natural or synthetic nucleic acid bases
including adenine, guanine, cytosine, thymine and uracil and
substituted derivatives of these bases. Compounds of the present
invention may also contain certain modifications of the
ribofuranosyl moiety.
The present invention also relates to methods for inhibiting the
growth or replication of HBV comprising exposing the virus to an
inhibitory effective amount or concentration of at least one of the
disclosed L-2',3'-dideoxynucleoside analogs. This method may be
used in comparison tests such as assays for determining the
activities of related anti-HBV compounds as well for determining
the susceptibility of a patient's HBV infection to one of the
compounds according to the present invention. The present invention
may also be used in treating viral infections.
The present invention also relates to a method for inhibiting the
growth or replication of HIV comprising exposing the virus to an
inhibitory effective amount or concentration of
1-(2,3-dideoxy-beta-L-ribofuranosyl)-5-fluorocytosine. This method
may be used in comparison tests such as assays for determining the
activities of related anti-HIV compounds as well for determining
the susceptibility of a patient's HIV infection to the compound.
The present invention may also be used in treating viral
infections.
The therapeutic aspect according to the present invention relates
to methods for treating retroviral infections in animal or human
patients, in particular, HBV or HIV infections in humans comprising
administering anti-viral effective amounts of the compounds
according to the present invention to inhibit the growth or
replication of the viruses in the animal or human patient being
treated.
Pharmaceutical compositions based upon these novel chemical
compounds comprise the above-described compounds in a
therapeutically effective amount for treating a viral, preferably a
Hepatitis B viral, and in certain instances, a HIV infection,
optionally in combination with a pharmaceutically acceptable
additive, carrier or excipient.
Certain of the compounds, in pharmaceutical dosage form, may be
used as prophylactic agents for inhibiting the growth or
replication of the viral infection. These may be particularly
appropriate as anti-HBV or anti-HIV agents. In certain
pharmaceutical dosage forms, the pro-drug form of the compounds
according to the present invention are preferred.
While not being limited by way of theory, it is believed that the
compounds according to the present invention induce their
inhibitory effect on the growth or replication of HBV or HIV by
functioning as anti-metabolites of the reverse transcriptase enzyme
of HBV and HIV.
The compounds according to the present invention are produced by
synthetic methods which are readily known to those of ordinary
skill in the art and include various chemical synthetic
methods.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1-11 (Schemes 1-11) depict the synthetic chemical steps which
are used to synthesize the compounds according to the present
invention. Schemes pertaining to the synthesis of a particular
composition are referenced in the examples set forth herein.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
The following definitions will be used throughout the specification
to describe the present invention.
The term "dideoxy" is used throughout the specification to describe
ribofuranosyl moieties which contain hydrogens rather than
hydroxyls at the 2' and 3' positions of the sugar in the present
compounds.
The term "didehydro" is used throughout the specification to
describe ribofuranosyl moieties which contain a double bond. For
example, 2',3'-didehydro refers to a ribofuranosyl moiety
containing a double bond between the 2' and 3' carbons of the
sugar.
The term "inhibitory effective concentration" or "inhibitory
effective amount" is used throughout the specification to describe
concentrations or amounts of compounds according to the present
invention which substantially or appreciably inhibit the growth or
replication of susceptible viruses, especially including HBV or
HIV.
The term "therapeutic effective amount" is used throughout the
specification to describe concentrations or amounts of compounds
according to the present invention which are therapeutically
effective in treating retroviral infections, and in particular, HBV
or HIV infections in humans.
The term "L-configuration" is used throughout the specification to
describe the chemical configuration of the dideoxyribofuranosyl
moiety of compounds according to the present invention. The
L-configuration of the sugar moiety of compounds of the present
invention contrasts with the D-configuration of ribose sugar
moieties of the naturally occurring nucleosides cytidine,
adenosine, thymidine, guanosine and uridine.
The present invention relates to the surprising discovery that
certain dideoxynucleoside analogs which contain a dideoxy
ribofuranosyl moiety having an L-configuration (as opposed to the
naturally occurring D-configuration) exhibit unexpected activity
against Hepatitis B virus (HBV). In particular, the compounds
according to the present invention show potent inhibition of the
replication of the virus in combination with very low toxicity to
the host cells (i.e., animal or human tissue).
The present invention also relates to the unexpected discovery that
the compound 1-(2,3-dideoxy-beta-L-ribofuranosyl)-5-fluorocytosine
(.beta.-L-FddC) is an extremely effective anti-HIV agent exhibiting
relatively low toxicity, especially compared to
1-(2,3-dideoxy-beta-D-ribofuranosyl)cytosine (dideoxycytidine or
ddC) which is presently used as one of the most effective anti-HIV
compounds presently available. That .beta.-L-FddC would exhibit
such exception anti-HIV activity and relatively limited toxicity to
the host is an unexpected result, especially when compared to the
anti-HIV activity of similar compounds.
The present invention relates to a first group of compounds
according to the structure: ##STR1## where Y is ##STR2## and R is
F, Cl, Br, I or CH.sub.3.
In this first group of compounds, R is preferably H or F.
The present invention also relates to a second group of compounds
according to the structure: ##STR3## where R is H, F, Cl, Br, I or
CH.sub.3.
In this second group of compounds, R is preferably H or F, most
preferably H.
The present invention also relates to a third group of compounds
according to the structure: ##STR4## where X is H, F, Cl, Br, I,
CH.sub.3, --C.tbd.CH, --HC.dbd.CH.sub.2 or ##STR5##
In this third group of compounds, X is preferably H, F or CH.sub.3,
most preferably CH.sub.3.
The present invention also relates to a fourth group of compounds
according to the structure: ##STR6## whereB is ##STR7## R' is H or
CH.sub.3 ; R" is H or CH.sub.3 ;
Y" is H, F, Br, Cl or NH.sub.2 when R' and R" are H and
Y" is H when at least one of R' or R" is CH.sub.3 ;
and Z is H or NH.sub.2.
In this fourth group of compounds according to the present
invention, R' and R" are preferably H and Y" is preferably H or R,
most preferably H. Z is preferably NH.sub.2.
The present invention also relates to compounds having the
structures: ##STR8## where T is F, Cl, Br or NH.sub.2 ; ##STR9##
where W is H or NH.sub.2.
In a first method aspect, the present invention relates to a method
for inhibiting the growth or replication of Hepatitis B virus
comprising exposing the virus to an inhibitory effective
concentration of a compound according to the structure: ##STR10##
where Y is ##STR11## and R is H, F, Cl, Br, I or CH.sub.3.
In this first method aspect, R is preferably H or F.
A second method aspect for inhibiting the growth or replication of
Hepatitis B virus according to the present invention comprises
exposing the virus to an inhibitory effective concentration of a
compound according to the structure: ##STR12## where X is H, F, Cl,
Br, I, CH.sub.3, --C.tbd.CH, --HC.dbd.CH.sub.2 or ##STR13##
In this second method aspect, X is H, F or CH.sub.3, most
preferably CH.sub.3.
A third method aspect for inhibiting the growth or replication of
Hepatitis B virus according to the present invention comprises
exposing the virus to an inhibitory effective concentration of a
compound according to the structure: ##STR14## where B is ##STR15##
R' is H or CH.sub.3 ; R" is H or CH.sub.3 ;
Y" is H, F, Cl, Br or NH.sub.2 when R' and R" are H and
Y" is H when at least one of R' or R" is CH.sub.3 ;
and Z is H or NH.sub.2.
In this third method aspect of the present invention, R' and R" are
preferably H and Z is preferably NH.sub.2.
A fourth method aspect for inhibiting the growth or replication of
Hepatitis B virus according to the present invention comprises
exposing the virus to an inhibitory concentration of a compound
according to the structure: ##STR16## where T is H, F, Cl, Br or
NH.sub.2.
In this fourth method T is preferably H or F, most preferably
H.
A fifth method aspect for inhibiting the growth or replication of
Hepatitis B virus according to the present invention comprises
exposing the virus to an inhibitory concentration of a compound
according to the structure: ##STR17## where W is H or NH.sub.2.
A sixth method aspect according to the present invention relates to
the inhibition of the growth or replication of human
immunodeficiency virus according to the present invention
comprising exposing the virus to an inhibitory concentration of a
compound according to the structure: ##STR18## where Y is ##STR19##
and R is F.
The present invention is also directed to a method for treating a
patient suffering from an infection caused by the human
immunodeficiency virus comprising administering to said patient a
therepeutically effective concentration of a compound according to
the structure: ##STR20## where Y is ##STR21## and R is F.
The compounds according to the present invention are primarily
useful for their anti-retroviral activity and in particular, their
anti-HBV or anti-HIV activity. The present compounds may also be
useful for their biological activity as antifungal or antimicrobial
agents. In addition, these compositions may also find use as
intermediates in the chemical synthesis of other nucleoside or
nucleotide analogs which are, in turn, useful as therapeutic agents
or for other purposes. Preferably, these compositions find use as
novel anti-HBV agents and, in addition, in the case of
.beta.-L-FddC, also as a novel anti-HIV agent.
In general, the most preferred anti-viral, especially anti-HBV or
anti-HIV compounds, according to the present invention include
those which are less cytotoxic to the host cells and more active to
the targeted virus. Certain of the compounds, in pharmaceutical
dosage form, may be used as prophylactic agents. These may be
particularly appropriate as antiviral agents, and in particular,
anti-HBV or anti-HIV agents. Because of its very low toxicity to
the patient, .beta.-L-FddC is an especially effective
anti-propylactic compound for inhibiting HIV and preventing
AIDS.
The compounds according to the present invention are produced by
synthetic methods which are readily known to those of ordinary
skill in the art and include various chemical synthetic methods as
elaborated in significantly more detail in the Examples which
follow. In general, compounds according to the present invention
are synthesized by condensing a previously synthesized nucleoside
base onto the appropriate sugar synthon which will ultimately give
rise to a nucleoside analog having the desired dideoxyribofuranosyl
moiety of L-configuration. In certain instances, the synthetic
pathway may deviate from the general synthetic pathway for a
specific nucleoside analog (for example, in the case of
1-(2,3-dideoxy-beta-L-ribofuranosyl)cytosine and
1-(2,3-dideoxy-beta-L-ribofuranosyl)uracil as set forth in Example
1 and Scheme 3.
During chemical synthesis of the various compositions according to
the present invention, one of ordinary skill in the art will be
able to practice the present invention without undue
experimentation. In particular, one of ordinary skill in the art
will recognize the various steps that should be performed to
introduce a particular substituent at the desired position of the
base or a substituent at the desired position on the sugar moiety.
In addition, chemical steps which are taken to "protect" functional
groups such as hydroxyl or amino groups, among others, as well as
"de-protect" these same functional groups, will be recognized as
appropriate within the circumstances of the syntheses.
The therapeutic aspect according to the present invention relates
to methods for treating retroviral infections in animal or human
patients, in particular, HBV or HIV infections in humans comprising
administering anti-viral effective amounts of the compounds
according to the present invention to inhibit the growth or
replication of the viruses in the animal or human patient being
treated.
Pharmaceutical compositions based upon these novel chemical
compounds comprise the above-described compounds in a
therapeutically effective amount for treating a viral, preferably a
Hepatitis B viral or HIV infection, optionally in combination with
a pharmaceutically acceptable additive, carrier or excipient. One
of ordinary skill in the art will recognize that a therapeutically
effective amount will vary with the infection or condition to be
treated, its severity, the treatment regimen to be employed, the
pharmacokinetics of the agent used, as well as the patient (animal
or human) treated.
In the pharmaceutical aspect according to the present invention,
the compound according to the present invention is formulated
preferably in admixture with a pharmaceutically acceptable carrier.
In general, it is preferable to administer the pharmaceutical
composition in orally-administrable form, but certain formulations
may be administered via a parenteral, intravenous, intramuscular,
transdermal, buccal, subcutaneous, suppository or other route.
Intravenous and intramuscular formulations are preferably
administered in sterile saline. Of course, one of ordinary skill in
the art may modify the formulations within the teachings of the
specification to provide numerous formulations for a particular
route of administration without rendering the compositions of the
present invention unstable or compromising their therapeutic
activity. In particular, the modification of the present compounds
to render them more soluble in water or other vehicle, for example,
may be easily accomplished by minor modifications (salt
formulation, esterification, etc.) which are well within the
ordinary skill in the art. It is also well within the routineer's
skill to modify the route of administration and dosage regimen of a
particular compound in order to manage the pharmacokinetics of the
present compounds for maximum beneficial effect in patients.
In certain pharmaceutical dosage forms, the pro-drug form of the
compounds, especially including acylated (acetylated or other)
derivatives, pyridine esters and various salt forms of the present
compounds are preferred. One of ordinary skill in the art will
recognize how to readily modify the present compounds to pro-drug
forms to facilitate delivery of active compounds to a targeted site
within the host organism or patient. The routineer also will take
advantage of favorable pharmacokinetic parameters of the pro-drug
forms, where applicable, in delivering the present compounds to a
targeted site within the host organism or patient to maximize the
intended effect of the compound.
The amount of compound included within therapeutically active
formulations according to the present invention is an effective
amount for treating the infection or condition, in its most
preferred embodiment, an HBV infection, or in the case of
.beta.-L-FddC, an HIV infection. In general, a therapeutically
effective amount of the present compound in dosage form usually
ranges from slightly less than about 1 mg./kg. to about 25 mg./kg.
of the patient or considerably more, depending upon the compound
used, the condition or infection treated and the route of
administration. In the case of HBV infections, the compound is
preferably administered in amounts ranging from about 1 mg/kg to
about 25 mg/kg. In the case of the use of .beta.-L-FddC as an
anti-HIV agent, the compound is preferably administered in an
amount ranging from about 1 mg/kg to about 25 mg/kg, depending upon
the pharmacokinetics of the agent in the patient. This dosage range
generally produces effective blood level concentrations of active
compound ranging from about 0.04 to about 100 micrograms/cc of
blood in the patient.
Administration of the active compound may range from continuous
(intravenous drip) to several oral administrations per day (for
example, Q.I.D.) and may include oral, topical, parenteral,
intramuscular, intravenous, sub-cutaneous, transdermal (which may
include a penetration enhancement agent), buccal and suppository
administration, among other routes of administration.
To prepare the pharmaceutical compositions according to the present
invention, a therapeutically effective amount of one or more of the
compounds according to the present invention is preferably
intimately admixed with a pharmaceutically acceptable carrier
according to conventional pharmaceutical compounding techniques to
produce a dose. A carrier may take a wide variety of forms
depending on the form of preparation desired for administration,
e.g., oral or parenteral. In preparing pharmaceutical compositions
in oral dosage form, any of the usual pharmaceutical media may be
used. Thus, for liquid oral preparations such as suspensions,
elixirs and solutions, suitable carriers and additives including
water, glycols, oils, alcohols, flavouring agents, preservatives,
colouring agents and the like may be used. For solid oral
preparations such as powders, tablets, capsules, and for solid
preparations such as suppositories, suitable carriers and additives
including starches, sugar carriers, such as dextrose, mannitol,
lactose and related carriers, diluents, granulating agents,
lubricants, binders, disintegrating agents and the like may be
used. If desired, the tablets or capsules may be enteric-coated or
sustained release by standard techniques.
For parenteral formulations, the carrier will usually comprise
sterile water or aqueous sodium chloride solution, though other
ingredients, including those which aid dispersion, also may be
included. Of course, where sterile water is to be used and
maintained as sterile, the compositions and carriers must also be
sterilized. Injectable suspensions may also be prepared, in which
case appropriate liquid carriers, suspending agents and the like
may be employed.
In particularly preferred embodiments according to the present
invention, the compounds and compositions are used to treat
retroviral infections of mammals and in particular humans. In its
most preferred embodiment, the compounds are used to treat HBV
infections, including chronic HBV infection. The comound
.beta.-L-FddC is effectively used to treat HIV infections,
including AIDS. Generally, to treat HBV or HIV infections, the
compositions preferably will be administered in oral dosage form in
amounts ranging from about 250 micrograms up to about 500 mg. or
more up to four times a day. The present compounds are preferably
administered orally, but may be administered parenterally,
topically or in suppository form.
The compounds according to the present invention, because of their
unexpectedly low toxicity to host cells, may advantageously be
employed prophylactically to prevent infection or to prevent the
occurrence of clinical symptoms associated with the viral
infection. Thus, the present invention also encompasses methods for
the therapeutic or prophylactic treatment of viral infections, and
in particular HBV or HIV infections. This prophylactic method
comprises administering to a patient in need of such treatment an
amount of a compound according to the present invention effective
for alleviating, and/or preventing the viral infection. In the
prophylactic treatment according to the present invention, it is
preferred that the antiviral compound utilized should be as low in
toxicity and preferably non-toxic to the patient. It is
particularly preferred in this aspect of the present invention that
the compound which is used should be maximally effective against
the virus and should exhibit a minimum of toxicity to the patient.
In the case of .beta.-L-FddC, this compound may be administered
within the same dosage range for therapeutic treatment (i.e., about
250 micrograms up to about 500 mg. from one to four times per day
for an oral dosage form) as a prophylactic agent to prevent the
rapid proliferation of HIV or alternatively, to prolong the onset
of AIDS in a patient.
In addition, compounds according to the present invention may be
administered alone or in combination with other agents, especially
including other compounds of the present invention. Certain
compounds according to the present invention may be effective for
enhancing the biological activity of certain agents according to
the present invention by reducing the metabolism or inactivation of
other compounds and as such, are co-administered for this intended
effect. In the case of .beta.-L-FddC, this compound may be
effectively combined with any one or more of the standard anti-HIV
agents which are presently utilized including AZT, DDC, and DDI,
among others.
In a particularly preferred pharmaceutical composition and method
for treating HBV infections, an inhibitory effective amount of
1-(2,3-dideoxy-beta-L-ribofuranosyl)cytosine and/or
1-(2,3-dideoxy-beta-L-ribofuranosyl)5-fluoro-cytosine is
administered to a patient suffering from an HBV infection to
alleviate the symptoms of such infection.
In a particularly preferred pharmaceutical composition and method
for treating HIV infections, an inhibitory effective amount of
1-(2,3-dideoxy-beta-L-ribofuranosyl)5-fluorocytosine is
administered to a patient suffering from an HIV infection and/or
AIDS to alleviate the symptoms of such infection.
While not being limited by way of theory, it is believed that the
compounds according to the present invention primarily induce their
inhibitory effect on the growth or replication of HBV or HIV by
functioning as anti-metabolites of the reverse transcriptase enzyme
of the virus.
The present invention is now described, purely by way of
illustration, in the following examples. It will be understood by
one of ordinary skill in the art that these examples are in no way
limiting and that variations of detail can be made without
departing from the spirit and scope of the present invention.
EXAMPLES
I. Chemical Synthesis of L-2'3'-dideoxynucleoside Analogs
Examples 1-9
In general, compounds of the present invention are synthesized
according to the chemical synthetic method described hereinbelow.
The synthetic chemical methodology employed to synthesize the
present compounds represents modifications of literature
procedures. The references from which a related chemical reaction
have been modified to produce the present compounds are set forth
in the examples, below.
Melting points were determined using a MelTemp apparatus and are
uncorrected. Proton NMR spectra were recorded on a Varian EM390 or
Bruker WM 250 instrument and reported as ppm (delta) downfield from
(CH.sub.3).sub.4 Si. Ultraviolet spectra were recorded on a Beckman
25 spectrophotometer. Analytical thin-layer chromotography (TLC)
was done using Merck EM Silica Gel 60 F.sub.254 precoated sheets.
Column chromatography employed Merck EM silica gel using standard
organic solvents (CH.sub.2 Cl.sub.2 /MeOH or CH.sub.2 Cl.sub.2
/EtOAC varying in volume/volume ratio) unless otherwise indicated
primarily to separate the alpha and beta anomeric mixtures.
EXAMPLE 1
Synthesis of 1-(2,3-Dideozy-beta-L-ribofuranosyl)cytosine,
1-(2,3-Dideoxy-beta-L-ribofuranosyl)-5-fluoro-, -5-bromo-,
-5-chloro-, -5-iodo- and -5-methylcytosine
The methodology of Taniguchi et al. (Tetrahedron, 30, 3532, 1974)
and Farina et al. (Tetrahedron Lett., 29, 1239, 1988) for the
syntheses of D-ribose derivatives provided a model for our
synthetic approach to the syntheses of the corresponding L-ribose
derivatives. Nitrous acid deamination of D-glutamic acid (1) gave
lactone 2, which was then converted to the corresponding ester 3 by
treatment of compound 2, with ethanol and catalytic amount of
p-toluenesulfonic acid (See Scheme 1). Reduction of compound 3 with
NaBH.sub.4 in ethanol gave (R)-4-(hydroxymethyl)-4-butyrolactone
(4). Protection of the hydroxy group of compound 4 with
tert-butyldimethylsilyl chloride in methylene chloride using
imidazole as catalyst produced
(R)-4-{[(tert-butyldimethylsilyl)oxy]methyl}-4-butyrolactone (5),
which was then converted to the corresponding lactol 6 by reduction
with diisobutyaluminum hydride (DIBAL) in toluene at -78.degree. C.
Acetylation of 6 with acetic anhydride and triethylamine afforded
the key sugar intermediate,
1-O-acetyl-5-O-(tert-butyldimethylsilyl)-2,3-dideoxy-L-ribofuranose
(7) as a mixture of alpha and beta anomers: MS, m/e 231 (M.sup.+
-CH.sub.3 CO), 215 (M.sup.+ -CH.sub.3 COO); NMR(CDCl.sub.3)) delta
0.10 (s, 6H, SiMe.sub.2), 0.95 (s, 9H, tert-butyl), 1.85-2.15 (m,
7H, CH.sub.2 CH.sub.2 and COCH.sub.3), 3.50-3.65 (M, 2H, 5-H),
4.10-4.30 (m, 1H, 4-H), 6.20-6.30 (m, 1H, 1-H).
Uracil, 5-fluoro, 5-bromo-, 5-chloro- and 5-iodouracil as well as
thymine were coupled with acetate 7 by the methodology of Okabe et
al. (J. Org. Chem., 53, 4780, 1988) with minor modifications.
Silylated 5-fluorouracil, prepared from 5-fluorouracil, (4.3 g, 33
mmol) was reacted with acetate 7 (8.3 g, 30 mmol) and ethylaluminum
dichloride (16.7 mL of a 1.8M solution in toluene, 30 mmol) in
methylene chloride at room temperature for 3 hrs. to give 8.5 g
(83%) of 1-{5-O(tert-butyldimethylsilyl)-2,3-dideoxy-alpha,
beta,-L-ribofuranosyl]-5-fluorouracil (8,R.dbd.F) as a 2:3
alpha/beta anomeric mixture. The alpha and beta anomers were
separated by silica gel chromatography. The beta anomer (9): NMR
(CDCl.sub.3) delta 0.10 (s, 6H, SiMe.sub.2), 0.95 (s, 9H,
tert-butyl), 1.80-2.45 (m, 4H, 2'-H and 3'-H), 3.50-3.70 (m, 1H,
4'-H), 3.95-4.15 (m, 2H, 5'-H), 5.90-6.05 (m, 1H, 1'-H), 8.10-8.20
(d, 1H, 6-H), 9.30-9.50 (br, 1H, NH, D.sub.2 O exchangeable); the
alpha isomer: NMR (CDCl.sub.3) delta 0.10 (s, 6H, SiMe.sub.2), 0.95
(s, 9H, tert-butyl), 1.90-2.55 (m, 4H, 2'-H and 3'-H), 3.60-3.65
(m, 2H, 5'-H), 4.30-4.50 (m, 1H, 4'-H), 5.90-6.05 (m, 1H, 1'-H),
7.30-7.40 (d, 1H, 6-H), 9.00-9.30 (br, 1H, NH, D.sub.2 O
exchangeable). Treatment of the beta anomer (9, 3 g, 8.7 mmol) with
4-chlorophenyl phosphorodichloridate (6.2 mL, 37.8 mmol) and
1,2,4-triazole (7.9 g, 114 mmol) in anhydrous pyridine (60 mL) at
room temperature yielded the 4-triazolylpyrimidinone derivative 10.
The crude product 10 was treated with a mixture of ammonium
hydroxide/dioxane (1.:3, v/v) to afford the 2',3'-dideoxycytidine
drivative 11 (1.2 g, 40%), which was then deblocked by reaction
with tetra-n-butylammonium fluoride in THF at room temperature for
20 min to afford the target compound
1-(2,3-dideoxy-beta-L-ribofuranosyl)-5-fluorocytosine (12, R.dbd.F,
L-FDDC): mp 147.degree.-149.degree. C.; NMR (DMSO-d.sub.6) delta
1.85-2.35 (m, 4H, 2'-H and 3'-H), 3.60-3.82 (m, 2H, 5'-H), 4.25 (m,
1H, 4'-H), 5.15 (t, 1H, 5'-OH, D.sub.2 O exchangeable), 5.95-6.15
(m, 1H, 1'-H), 7.45 (br s, 2H, 4-NH.sub.2, D.sub.2 O exchangeable),
8.22 (d, 1H, 6-H).
To synthesize 1-(2,3-dideoxy-beta-L-ribofuranosyl)cytosine,
1(2,3-dideoxy-beta-L-ribofuranosyl)-5-bromo-, -5-chloro-, -5-iodo-,
or -5-methylcytosine, the analogous procedure used to synthesize
the 5-fluoro derivative was employed. For the coupling reaction,
the corresponding silylated 5-bromo-, -5-chloro-, -5-iodo-, or
-5-methylcytosine was used instead of 5-fluorouracil. All other
steps are analogous to those for the synthesis of the 5-fluoro
derivatitve 12 (R.dbd.F).
Treatment of compound 9 with tetra-n-butylammonium fluoride in THF
gave the corresponding uracil derivative 13.
Compound 12 (R.dbd.H,F,Cl,I and CH.sub.3) was also synthesized by
an alternative methodology (See Scheme 2), by which the silylated
compound 15 (R.dbd.H,F,Cl,I and CH.sub.3), prepared from the
corresponding cytosine (14, R.dbd.H) and its derivatives 14
(R.dbd.F,Cl,I and CH.sub.3), were directly coupled with acetate 7,
followed by separation of the alpha and beta anomers 16 and removal
of the protecting group.
Compound 12 (R.dbd.H) was also synthesized by a stereospecific
approach (See Scheme 3), in which the possibility of producing the
alpha anomer was eliminated. O-2,2'-Anhydrouridine 19 was prepared
by the method of Holy (Collection Czechoslov. Chem. Commun., 37,
4072, 1972) from L-arabinose (17) via the intermediate oxazoline
derivative 18. Treatment of compound 19 with
tert-butyldimethylsilyl chloride in pyridine gave the protected
chloro derivative,
1-[5-O(-tert-butyldimethylsilyl)-2-chloro-2-deoxy-beta
-L-ribofuranosyl]uracil (20, R.dbd.H). Conversion of compound 20 to
the corresponding 2',3'-unsaturated nucleoside 22 was achieved by
previously developed methodology (Lin et al., Tetrahedron Lett.,
31, 3829, 1991). Treatment of compound 20 with phenyl
chlorothionocarbonate and 4-dimethylaminopyridine in acetonitrile
under nitrogen at room temperature yielded the
2'-chloro-3'-O-phenoxythiocarbonyl derivative 21, which has two
different vicinal groups at the 2'- and 3'-positions. Reduction of
compound 21 with tri-n-butyltin hydride and azobisisobutyronitrile
(AIBN) in dry toluene at 60.degree.-70.degree. C. for 4 h produced
the 2',3'-unsaturated derivative 22 as a foam: NMR (CDCl.sub.3)
delta 0.10 (s, 6H, SiMe.sub.2), 0.95 (s, 9H, tert-butyl), 3.90 (m,
2H, 5'-H), 4.90 (m, 1H, 4'-H), 5.65-5.75 (d, 1H, 5-H), 5.80-5.90
(d, 1H, 3'-H), 6.25-6.35 (d, 1H, 2'-H), 7.05-7.10 (m, 1H, 1'-H),
7.75-7.85 (d, 1H, 6H), 9.55 (s, 1H, --NH, D.sub.2 O exchangeable).
Catalytic hydrogenation of compound 22, followed by treatment with
4-chlorophenyl phosphorodichloridate and 1,2,4-triazole yielded the
4-triazolylpyrimidinone derivative 24, which was then converted to
the desired 1-(2,3-dideoxy-beta-L-ribofuranosy)cytosine 12
(R.dbd.H, L-DDC) by treatment of 24 with NH.sub.4 OH/dioxane,
followed by deblocking of the 5'- protecting group as previously
described.
Compound 12 (R.dbd.H, L-DDC): mp 194.degree.-196.degree. C.; .sup.1
H NMR (DMSO-d.sub.6) 1.74-2.24 (m, 4-H, 2'-H and 3'-H), 3.49-3.65
(m, 2H, 5'-H), 3.98-3.99 (m, 1H, 4'-H), 4.96-5.00 (t, 1H, 5'-OH,
D2O exchangeable), 5.65-5.68 (d, 1 H, 5-H), 5.85-5.93 (m, 1H,
1'-H), 7.01-7.06 (m 2H, --NH2, D2O exchangeable), 7.87-7.90 (d, 1H,
6-H).
Treatment of compound 23 with tetra-n-butylammonium fluoride in THF
gave the corresponding uracil derivative 13 (R.dbd.H): .sup.1 HNMR
(DMSO-d.sub.6) delta 1.80-2.05 (m, 4-H, 2'-H and 3'-H), 3.45-3.60
(m, 2H, 5'-H), 3.85-4.05 (m, 1H, 4'-H), 4.85-5.00 (t, 1H, 5'-OH,
D.sub.2 O exchangeable), 5.45-5.55 (d, 1H, 5-H), 5.80-6.00 (m, 1H,
1'H), 7.80-7.90 (d, 1H, 6-H), 11.10 (s, 1H, NH, D2O
exchangeable).
EXAMPLE 2
2',3'-Dideoxy-, 2',3'-Dideoxy-N-methyl- and
2',3'-Dideoxy-N,N-dimethyl-beta-L-adenosine, and
2',3'-Dideoxy-L-inosine and 2',3'-Dideoxy-beta-L-guanosine
The synthesis of 2',3'-dideoxy-, 2'3'-dideoxy-6-N-methyl-, and
2',3'-dideoxy-N,N-dimethyl-beta-L-adenosine, and
2',3'-dideoxy-beta-L-inosine and 2',3'-dideoxy-beta-L-guanosine
(See Scheme 4) was based on the methodology reported by Fujimori et
al. (Nucleoside & Nucleotides, 11, 341, 1992) for the synthesis
of purine 2'-deoxynucleosides. Treatment of 6-chloropurine with NaH
(60% in oil, washed with n-hexane) and acetate 7 in anhydrous
acetonitrile under argon produced
6-chloro-9-[(5-O-tert-butyldimethylsilyl)-2,3-dideoxy-beta-L-erythro-pento
furanosyl]purine (26) together with the corresponding N-7 glycosyl
isomer, which was separated by silica gel chromatography.
Subsequent treatment of compound 26 with NH.sub.3 /CH.sub.3 OH,
CH.sub.3 NH.sub.2 /CH.sub.3 OH, or (CH.sub.3).sub.2 NH/CH.sub.3 OH
at elevated temperature, followed by deprotection with
tetra-n-butylammonium fluoride in THF afforded
2',3'-dideoxy-L-adenosine (27, R.dbd.R'.dbd.H),
2',3'-dideoxy-N-methyl-beta-L-adenosine (27, R.dbd.H,
R'.dbd.CH.sub.3,) and 2'3'-dideoxy-N,N-dimethyl-beta-L-adenosine
(27, R.dbd.R'.dbd.CH.sub.3), respectively. Treatment of compound 26
with tetra-n-butylammonium fluoride in THF, followed by alkaline
hydrolysis of the deblocking nucleoside (28) with 2N KOH/dioxane
(1:1, v/v) gave 2'3'-dideoxy-L-inosine (29). Similarly, treatment
of 2-amino-6-chloropurine with NaH (60% in oil, washed with
n-hexane) and acetate 7 in anhydrous acetonitrile under argon
afforded
2-amino-6-chloro-9-[(5-O-tert-butyldimethylsilyl)-2,3-dideoxy-beta-L-eryth
ro-pentofuranosyl]purine (30). Conversion of compound 30 to the
final product, 2'3'-dideoxy-beta-L-guanosine (32) was achieved via
the intermediate 31 by deblocking with tetra-n-butylammonium
fluoride in THF and alkaline hydrolysis with 2N KOH/dioxane
(1:1,v/v).
EXAMPLE 3
2-Chloro-, 2-Bromo-, 2-Amino-, and
2-Fluoro-2',3'-dideoxy-beta-L-adenosine
These compounds are synthesized as described in Scheme 5 by the
methodology employed in Example 2. 2,6-dichloropurine, prepred by
the method described by Elion and Hitching (J. Am. Chem. Soc., 78,
3508, 1956) was treated with NaH (60% in oil, washed with n-hexane)
and acetate 7 in anhydrous acetonitrile under argon to give
2,6-dichloro-9-[(5-O-tert-butyldimethylsilyl)-2,3-dideoxy-beta-L-erythrope
nto-furanosyl]purine (33) and the corresponding N-7 glycosyl
isomer, which was separated by silica gel chromatography. Treatment
of compound 33 with NH.sub.3 /CH.sub.3 OH at elevated temperature,
followed by deprotection with tetra-n-butylammonium fluoride in THF
afforded 2-chloro-2',3'-dideoxy-L-adenosine (34). Treatment of
dibromopurine with NaH (60% in oil, washed with n-hexane) and
acetate 7 in anhydrous acetonitrile under argon to produce
6-bromo-9-[(5-O-tert-butyldimethylsilyl)-2,3-dideoxy-beta-L-erythro-pentof
uranosyl]purine (31) together with the corresponding N-7 glycosyl
isomer, which was separated by silica gel chromatography.
Subsequent treatment of compound 31 with NH3/CH3OH at elevated
temperature, followed by deprotection with tetra-n-butylammonium
fluoride in THF afforded
6-bromo-(2,3-dideoxy-beta-L-erthro-pentofuranosyl)purine (41).
2,6-Bis(benzamido)purine, prepared by the method described by
Davoll and Lowy (J. Am. Chem. Soc., 73, 1650, 1951) was treated
with NaH (60% in oil, washed with n-hexane) and acetate 7 in
anydrous acetonitrile under argon produced
2,6-bis(benzamido)-9-[(5-O-tert-butyldimethylsilyl)-2,3-dideoxy-beta-L-ery
thro-pentofuranosyl]purine (37), which was then subsequently
deblocked by reaction with tetra-n-butylammonium fluoride in THF
and sodium ethoxide in ethanol to give
2-amino-2',3'-dideoxy-beta-L-adenosine (38). Treatment of compound
38 with sodium nitrite and 48-50% fluoroboric acid below
-10.degree. C. yielded 2-fluoro-2',3'-dideoxy-beta-L-adenosine
(39).
EXAMPLE 4
1-(2,3-Didehydro-dideoxy-beta-L-ribuofuranosyl)cytosine,
1-(2,3-Didehydro-dideoxy-beta-L-ribofuranosyl)-5-fluoro-,
-5-bromo-, -5-chloro-, -5-iodo-, and -5-methylcytosine
These compounds were synthesized as set forth in Scheme 6 by a
methdology developed for the syntheses of the related D-isomers
(Lin, et al., Biochem. Phamacol., 36, 311, 1987; Lin, et al.,
Organic Preparations and Procedures Intl., 22, 265, 1990).
Treatment of 2'-deoxy-L-uridine (40,R.dbd.H), was prepared by the
procedure of Holy (Collection Czechoslov. Chem. Commun., 37, 4072,
1972), with 2 equivalents of methanesulfonyl chloride in dry
pyridine at -5.degree.-0.degree. C. gave the 3',5' di-O-mesyl
derivative (41, R.dbd.H). Conversion of compound 41 (R.dbd.H) to
2'-deoxy-3',5'-epoxy-beta-L-uridine (43, R.dbd.H) via the
intermediate anhydronucleoside 42 (R.dbd.H) by treatment with 1N
NaOH according the procedure of Horwitz et al. (J Org. Chem., 32,
817, 1967). Treatment of compound 43 (R.dbd.H) with 1,2,4-triazole
and 4-chlorophenyl phosphorodichloridate in dry pyridine yielded
the 4-triazolylpyrimidinone 44 (R.dbd.H), which was then reacted
with NH.sub.4 OH/dioxane to give the cytidine derivative 45
(R.dbd.H). Treatment of compound 45 (R.dbd.H) with potassium
t-butoxide in DMSO afforded the final product
1-(2,3-didehydro-2,3-dideoxy-beta-L-ribofuranosyl)cytosine (46,
R.dbd.H): .sup.1 HNMR (DMSO-d.sub.6) delta 3.50 (m, 2H, 5'-H), 4.72
(m, 1H, 4'-H), 4.92 (br s, 1H, 5'-OH, D.sub.2 O exchangeable), 5.64
(d, 1H, 5-H), 5.83 (m, 1H, 3'-H), 6.30 (m, 1H, 2'-H), 6.85 (m, 1H,
1'-H), 7.09-7.15 (br d, 2H, 4-NH.sub.2, D.sub.2 O exchangeable),
7.64 (D, 1H, 6-H).
EXAMPLE 5
2',3'-Didehydro-2',3'-dideoxy-beta-L-adenosine
2',3'-Didehydro-2',3'-dideoxy-beta-L-adenosine (51) was synthesized
as shown in Scheme 7 by the methodology of Barton et al.
(Tetrahedron, 49, 2793, 1993) and Chu et al. (J. Org. Chem., 54,
2217, 1989) for the preparation of the D-isomer. Treatment of
L-adenosine (47) with tert-butyldimethlsilyl chloride and imidazole
in dry DMF with exclusion of moisture for 20 h gave
5'-O-(tert-butyldimethylsilyl)-beta-L-adenosine (48), which was
then reacted with CS.sub.2, 5N NaOH solution, and CH.sub.3 I in
DMSO to afford the 2',3'-O-bis(dithiocarbonate) derivative 49.
Deoxygenation of 49 with triethylsilane and benzoyl peroxide under
argon, followed by deprotection of the olefin derivative 50 with
tetra-n-butylammonium fluoride in THF afforded the final product
51.
Synthesis of the corresponding
2',3'-Didehydro-2',3'-dideoxy-beta-L-guanosine and
2',3'-Didehydro-2',3'-dideoxy-beta-L-inosine analogs followed the
same procedure as above, starting from L-guanosine and L-inosine
respectively.
EXAMPLE 6
1-(2,3-Dideoxy-4-thio-beta-L-ribofuranosyl)cytosine,
1-(2,3-Dideoxy-4-thio-beta-L-ribofuranosyl)-5-fluoro-, -5-chloro-,
-5-bromo-, -5-iodo-, and -5-methylcytosine
The methodology of Secrist et al. (J. Med. Chem. 35, 533, 1922) for
the synthesis of 2',3'-dideoxy-4'-thio-D-nucleosides provided a
useful example for our synthetic approach to the synthesis of
1-2',3'-dideoxy-4'-thio-beta-L-nucleoside analogs (See Scheme
8).
D-glutamic acid (1) was treated with sodium nitrite in hydrochloric
acid to produce (R)-1,4-butyrolactone-4-carboxylic acid (2).
Compound 2 was then reduced by borane-dimethyl sulfide complex in
THF to give the corresponding (R)-4-(hydroxymethyl)-4-butyrolactone
(4), which was subsequently treated with tert-butyldiphenylsilyl
chloride in methylene chloride using imidazole as a catalyst to
afford
(R)-5-O-tert-butyldiphenylsilyl-4-hydroxymethyl-1,4-butyrolactone
(53). The protected lactone 53 was opened with sodium hydroxide in
ethanol and then converted to the methyl ester of
5-[(tert-butyldiphenylsilyl)oxy]-4-(R)-hydroxypentanoic acid (54)
by reaction with dimethyl sulfate in dimethyl sulfoxide. Commpound
54 was transformed into the methyl ester of
5-[(tert-butyldiphenylsilyl)oxy]-4-(S)-iodopentanoic acid (55) by
treatment with triphenylphosphine, imidazole and iodine.
Displacement of the iodo group in compound 55 by thioacetate in
toluene occurred readily to give the methyl ester of
4-(R)-(acetylthio)-5-[(tert-butyldiphenylsilyl)oxy]pentanoic acid
(56). Compound 56 was then treated with 2 equivalents of
diisobutylaluminum hydride (DIBAL) in toluene to reductively
deprotect the sulfur and reduce the methyl ester to an aldehyde,
thereby producing the thiolactol via spontaneous cyclization. The
thiolactol was acetylated with acetic anhydride in pyridine to give
1-O-acetyl-5-O-(tert-butyldiphenylsilyl)-2,3-dideoxy-4-thio-L-ribofuranose
(57): .sup.1 HNMR (CDCl.sub.3) delta 7.67 (m, 4H, ArH), 7.40 (m,
6H, ArH), 6.10 (m, 1H, 1-H), 3.70 (m, 1H, 4-H), 3.52 (m, 2H, 5-H),
2.20 (m, 2H, CH.sub.2), 2.00 (2 s, 3H, CH.sub.3 CO--), 1.92 (m, 2H,
CH.sub.2), 1.08 (s, 9H, tert-butyl).
Cytosine, 5-fluorocytosine, and the other 5-substituted cytosine
derivatives were coupled with the acetate 57 by the methodology of
Vorbruggen and Bennua (J. Org. Chem., 39, 3654, 1974) with
modifications. A mixture of cytosine (0.42 g, 3.80 mmol),
hexamethyldisilazane (HMDS, 0.54 mL, 2.52 mmol),
chlorotrimethylsilane (TMSCl 1.48 mL, 11.6 mmol), potassium
nonafluorobutanesulfonate (3.08 g, 8.9 mmol), and the acetate 57
(1.04 g, 2.52 mmol) in dry acetonitrile was stirred at room
temperature overnight to afford 0.65 g (55%) of
1-[5-O-(tert-butyldiphenylsiyl)-2,3-dideoxy-4-thio-alpha,beta-L-ribofurano
syl]cytosine (58 X.dbd.H) as a 4:3 alpha/beta- mixture. The alpha
and beta anomers were separated by silica gel column
chromatography. Deprotection of 58 (beta anomer) afforded
1-(2,3-dideoxy-4-thio-beta-L-ribofuranosyl)cytosine (59 X.dbd.H) in
60% yield: MS m/e 228 (M.sup.+ +1); .sup.1 HNMR (DMSO-d.sub.6)
delta 8.05 (d, 1H, H-6), 7.08 (br d, 2H, NH.sub.2, D.sub.2 O
exchangeable), 6.10 (m, 1H, 1'-H), 5.70 (d, 1H, H-5), 5.20 (br d,
1H, 5'-OH, D.sub.2 O exchangeable), 3.58 (m, 1H, 5'-H), 3.52 (m,
2H, 4'-H and 5'-H), 2.20 (m, 1H, 2'-H), 2.04 (m 2H, 2'-H and 3'-H),
1.88 (m, 1H, 3'-H).
EXAMPLE 7
1-(2,3-Dideoxy-4-thio-beta-L-ribofuranosyl)-5-methyl-, -5-ethyl-,
-5-vinyl-, -5-bromovinyl-, -5-ethynyl-, -5-fluoro-, -5-chloro-,
-5-bromo-, -5-iodouracil, and
1-(2,3-Dideoxy-4-thio-beta-L-ribofuranosyl)uracil
Thymine, uracil, -5-ethyl-, -5-vinyl-, -5-bromovinyl-, -5-ethynyl-,
-5-fluoro-, -5-chloro-, -5-bromo-, -5-iodouracil, and other
5-substituted uracil derivatives were coupled with
1-O-acetyl-5-O-(tert-butyldiphenylsilyl)-2,3-dideoxy-4-thio-L-ribofuranose
(57) using the same procedure as described in Example 6 to give the
respective 5-subsituted pyrimidine nucleosides.
A mixture of the acetate 57 (1.40 g, 3.32 mmol), thymine (0.52 g,
4.20 mmol), HMDS (0.70 mL, 3.32 mmol), TMSCl (1.60 mL, 12.8 mmol)
and potassium nonafluorobutanesulfonate (3.50 g, 10.16 mmol) in dry
acetonitrile was stirred at 25.degree. C. overnight under nitrogen
to give
1-[5-O(tert-butyldiphenylsilyl)-2,3-dideoxy-4-thio-alpha,beta-L-ribofurano
syl]thymine (60, X.dbd.CH.sub.3) 1.18 g (74%) as a 4:3 alpha/beta
anomeric mixture. The alpha/beta anomers were separated by silica
gel column chromatography. Deprotection of beta-anomer 60 afforded
1-(2,3)-dideoxy-4-thio-beta-L-ribofuranosyl)thymine (61,
X.dbd.CH.sub.3) in 55% yield: Ms m/e 243 (m.sup.+ +1); .sup.1 HMR
(DMSO-d.sub.6) delta 11.5 (br s, 1H, NH), 7.74 (s, 1H, 6-H), 6.11
(m, 1H, 1'-H), 5.00 (t, 1-H, 5'-OH, D.sub.2 O exchangeable), 3.70
(m, 1-H, 4'-H), 3.65 (m, 2H, 5'-CH.sub.2) 2.20-1.80 (m, 4H,
2'-CH.sub.2 and 3'-CH.sub.2), 1.79 (s, 3H, 5-CH.sub.3).
EXAMPLE 8
2',3'-Dideoxy-4'-thio-beta-L-adenosine,
2',3'-Dideoxy-4'-thio-N-Methyl-beta-L-,
-N,N-dimethyl-beta-L-adenosine,
2',3'-Dideoxy-4'-thio-beta-L-inosine, and
2',3'-Dideoxy-4'-thio-beta-L-guanosine
2',3'-Dideoxy-4'-thio-beta-L-adenosine,
2'3'-Dideoxy-4'-thio-N-methyl-beta-L-adenosine,
2'3'-Dideoxy-4'-thio-N,N-dimethyl-beta-L-adenosine,
2',3'-Dideoxy-4'-thio-beta-L-inosine, and
2',3'-Dideoxy-4'-thio-beta-L-guanosine were synthesized by the
similar methodology of Secrist et al. (J. Med. Chem., 35, 533,
1992) for the syntheses of 2',3'-dideoxy-4'-thio-D-nucleosides.
Sugar 57 (4.3 g, 10.4 mmol) was coupled with 6-chloropurine (2.4 g,
15.6 mmol) in the presence of diethylaluminum chloride (5.9 mL,
10.6 mmol) in acetonitrile (150 mL) at 0.degree.-5.degree. C. for 2
h, by the procedure of Niedballa and Vobruggen (J. Org. Chem., 39,
3654 1974), to give 2.81 g (53%) of
9-[5-O-(tert-butyldiphenlsilyl)-2,3-dideoxy-4-thio-alpha,beta-L-ribofurano
syl]-6-chloropurine as a 1:1 alpha/beta anomeric mixture. The alpha
and beta anomers were separated by silica gel column
chromatography. The beta-anomer 62 was treated with saturated
ammonia/methanol and then deprotected with 1M tetrabutylammonium
fluoride and THF to afford 2',3'-dideoxy-4'-thio-beta-L-adenosine
(63, R'.dbd.R".dbd.H): .sup.1 HNMR (DMSO-d.sub.6) delta 8.30 (s,
1H, 2-H), 8.10 (s, 1 H, 8-H), 7.30 (s, 2H, NH.sub.2, D.sub.2 O
exchangeable), 6.12 (m, 1H, 1'-H), 5.11 (br s, 1H, 5'-OH, D.sub.2 O
exchangeable), 3.70 (m, 3H, 5'-CH.sub.2, 4'-H), 2.42 (m, 2H,
2'-CH.sub.2), 2.13 (m, 1H, 3'-H), 2.00 (m, 1H, 3'-H).
Compound 62 was deprotected with 1M tetrabutylammonium fluoride and
THF to yield
9-(2,3-dideoxy-4'-thio-beta-L-ribofuranosyl)-6-chloropurine (64).
Alkaline hydrolysis (Fujimori, et al., Nucleosides &
Nucleosides, 11, 341, 1992) of the 6-chloro moiety in compound 64
afforded 2',3'-dideoxy-4'-thio-beta-L-inosine (65) in 45% yield: MS
m/e 253 (m.sup.+ +1); .sup.1 HNMR (D.sub.2 O) delta 8.52 (s, 1H,
2-H), 8.19 (s, 1H, 8-H), 6.10 (m, 1H, 1'-H), 3.94 (m, 1H, 5'-H),
3.75 (m, 2H, 5'-H, 4'-H), 2.52 (m, 2H, 2'-CH.sub.2), 2.30 (m, 1H,
3'-H) 1.92 (m, 1H, 3'-H).
2',3'-Dideoxy-4'-thio-beta-L-guanosine (68) was synthesized from
acetate (57) by the similar methodology as described for the
synthesis of compound 65: MS m/e 268 (m.sup.+ +1): .sup.1 HNMR
(DMSO-d.sub.6) delta 10.7 (br s, 1H, NH, D.sub.2 O exchangeable),
8.01 (s, 1H, 8-H), 6.55 (s, 2H, NH.sub.2, D.sub.2 O exchangeable),
5.90 (m, 1H, 1'-H), 5.09 (br s, 1 H, 5'-OH, D.sub.2 O
exchangeable), 3.70 (m, 1H, 4'-H), 3.50 (m, 2H, 5'-H), 2.36 (m, 2H,
2'-H), 2.17 (m, 1H, 3'-H), 1.93 (m, 1H, 3'-H).
EXAMPLE 9
2',3'-Dideoxy-4'-thio-2-chloro-beta-L-adenosine,
-2-amino-beta-L-adenosine, -2-fluoro-beta-L-adenosine,
-2-chloro-N-methyl-beta-L-adenosine,
-2-chloro-N,N-dimethyl-beta-L-adenosine, -2-bromo-beta-L-adenosine,
-2-bromo-N-methyl-beta-L-adenosine and -2-bromo-N,N-dimethyl-beta
-L-adenosine
2',3'-Dideoxy-4'-thio-2-chloro-beta-L-adenosine,
2-amino-beta-L-adenosine, -2-fluoro-beta-L-adenosine,
-2-chloro-N-methyl-beta-L-adenosine,
-2-chloro-N,N-dimethyl-beta-L-adenosine, -2-bromo-beta-L-adenosine,
-2-bromo-N-methyl-beta-L-adenosine and
-2-bromo-N,N-dimethyl-beta-L-adenosine and other beta-L-adenosine
derivatives were synthesized as set forth in Scheme 11.
9-[5-O-(tert-Butyldiphenylsilyl)-2,3-dideoxy-4-thio-beta-L-ribofuranosyl]-2
,6-dichloropurine (69) was synthesized from the acetate 57 and
2,6-dichloropurine by the similar methodology as described for the
synthesis of compound 62 in an approximate 2:3 alpha/beta anomer
ratio in 60% yield. The alpha and beta anomers were separated by
silica gel column chromatography. Compound 69 was treated with
saturated ammonia/methanol and then deprotected with 1M
tetrabutylammonium fluoride in THF to provide
2',3'-dideoxy-4'-thio-2-chloro-beta-L-adenosine (70
R'.dbd.R".dbd.H) in 52% yield: MS m/e 286 (m.sup.+ +1); .sup.1 HNMR
(DMSO-d.sub.6) delta 8.46 (s, 1H, 2-H), 7.82 (br s, 2H, NH.sub.2,
D.sub.2 O exchangeable), 6.10 (m, 1H, 1'-H), 5.10 (m, 1H, 5'-OH,
D.sub.2 O exchangeable), 3.74 (m, 1H, 4'-H), 3.60 (m, 2H, 5'-H),
2.42 (m, 2H, 2'-H), 2.13 (m, 1H, 3'-H), 2.02 (m, H, 3'-H).
2',3'-Dideoxy-4'thio-2-bromo-alpha,beta-L-adenosine (72,
R'.dbd.R".dbd.H) was synthesized by coupling the acetate 57 and
2,6-dibromopurine, followed by treatment of the respective amine by
the same methodology as described for the synthesis of compound
70.
Compound 69 was treated with lithium azide to give the diazido
nucleoside 73, which was then reduced with lithium aluminium
hydride (LAH) to produce
9-[5-O-(tert-butydiphenylsilyl)-2,3-dideoxy-4-thio-alpha,beta-L-ribofurano
syl]-2,6-diaminopurine (74). Compound 74 was deprotected with
tetrabutylammonium fluoride in THF to yield 2',3'-
dideoxy-4'-thio-2-amino-alpha,beta-L-adenosine (75), which was then
converted to 2',3'-dideoxy-4'-thio-2-fluoro-alpha,beta-L-adenosine
(76) by reaction with sodium nitrite and HBF.sub.4.
II. Biological Activity
A. Anti-HBV Effects
The biological activity of the present compounds was assessed as
described by Doong, S-L, et al., Proc. Natl. Acad. Sci. U.S.A 88,
8495-8499 (1991). The human hepatoma cell line carrying the HBV
(designated 2.2.15) kindly provided by Dr. G. Acs was used in the
study. Price, et al., Proc. Natl. Acad. Sci. U.S.A. 86, 8541
(1989). Briefly, six day-old cultures were treated with varying
concentrations of the drug in the culture medium (Minimum essential
medium with Earl's salts and 10% fetal bovine serum). The drug was
left in the culture medium for a period of 3 days after which
period the medium was aspirated and fresh medium containing the
same concentration(s) of the drug was added. At the end of the
subsequent 3 day period the culture medium was harvested. The
culture medium was processed for obtaining the virions by the
polyethylene glycol precipitation method (Doong, et al., supra).
Viral DNA thus recovered from the secreted particles was subjected
to Southern analysis. Inhibition of the viral replication was
determined by the comparison of the viral DNA from drug-treated
versus control cultures not treated with the drug.
To determine the cellular toxicity of the present compounds, the
T-lymphoblastoic cell line (CEM) was used. Cells were subjected to
varying concentrations of the drug(s) and cell numbers were
determined 3 days post treatment by the method described by Chen,
C-H and Cheng, Y-C J. Biol. Chem., 264, 11934 (1989).
Concentrations of the drug which would result in 50% killing of the
cell populations were determined from the plot generated by
representing cell numbers corresponding to the individual drug
concentrations.
The effects of the various drug concentrations on mitochondrial DNA
(mt DNA) was evaluated by the method described by Chen and Cheng,
supra. CEM cells treated with varying concentrations of the drug
were collected by centrifugation. After washing the cells with
phosphate buffered saline, cells were lysed by suspending the cells
in 10 mM Tris-HCl (pH 7.0) and repeating freeze thaw cycles. The
resulting cells were then subjected to RNase A treatment at a final
enzyme concentration of 10 ug/ml, followed by proteinase K
treatment (100 ug/ml) for 1 hour. The DNA thus obtained by this
procedure was then immobilized on nylon membrane after the addition
of 0.8 vol of NaI and boiling for 10 minutes. Hybridization of the
resulting DNA to a mt DNA specific probe was performed by following
the method of Doong, S-L, supra and autoradiography was also
performed. Quantitative estimates were obtained by scanning
densitometer. The blots were stripped of the mtDNA probe and
rehybridized to human Alu sequence probe to determine the amounts
of DNA for normalization and estimation of absolute amounts of the
mt DNA.
B. Anti-HIV Effects
Drug susceptibility assay for determining the effectiveness of the
compounds of the present invention against HIV in MT-2 cells is a
modification of the assay described in Mellors, et al., Molecular
Pharmacology, 41, 446 (1992). Drug-mediated inhibition of
virus-induced cell toxicity was measured by the A.sub.595 of MTT
([3-I 4,5-dimethyl thiazol-2-yl]-2,3-diphenyltetrazolium bromide)
(Sigma M-2128). Triplicate wells of a 96 well plate which contains
1.times.10.sup.4 MT2 cells (AIDS-repository) were infected with
HIV-1 (HTLV-IIIB Strain-R.C. Gallo) at a multiplicity of 0.01
TCID.sub.50 /cell. MT-2 cells in RPMI 1640 media supplemented with
10% dialysized fetal bovine and 100 ug/ml Kanamycin were infected
with virus and immediately added to serial dilution of the drug.
After 5 days, 20 ul of MTT dye (2.5 mg/ml in PBS) was added per
well. At the end of a four hour incubation period 150 ul of
acidified 2-propanol with NP-40 non-ionic detergent was added.
After the crystals of dye dissolve (usually 1-2 days), the plates
are read on a micro-plate reader. Using this MTT-dye reduction
method (as set forth by Larder, et al., Antimicrobial Agents and
Chemotherapy, 34, 436 (1990), the percentage of protection can be
calculated using the formula [(a-b/c-b).times.100] in which
a=A.sub.595 of drug treated cells, b is the number of non-drug
infected cells and c is the A.sub.595 of the non-drug infected
cells.
The ID.sub.50 values for anti-HIV activity of the compound
.beta.-L-FddC and other compounds are presented in Table 1,
below.
C. Results of Biological Testing
Analysis of the viral replication of HBV from the secreted
particles revealed that the DNA replication was efficiently
inhibited by both .beta.-L-ddC and .beta.-L-FddC. The ID.sub.50
concentration required to inhibit the viral replication by these
compounds was 0.01 uM. The cellular cytotoxicity of these compounds
as compared to ddC was also considerably less as evidenced by the
Table 1 set forth below. It is interesting to note that these
compounds have several fold higher activity against HBV with
minimal cellular effects, an unexpected result. ddC on the other
hand, was much more cytotoxic than either .beta.-L-ddC or
.beta.-L-FddC. In addition, ddC also was shown to exhibit
significant effects on host mitochonrial DNA. It is expected that
.beta.-L-ddC and .beta.-L-FddC would have significantly lower
adverse effects on the mitochondrial DNA than ddC as concentrations
as high as 100 uM of .beta.-L-ddC or .beta.-L-FddC were not
inhibitory in the assay. This result is particularly significant
inasmuch as ddC exhibits dose limiting toxicity in causing severe
neuropathy, a condition which is believed to be at least in part
caused by inhibition of host mitochondrial DNA. Based upon these
results, .beta.-L-ddC and .beta.-L-FddC are extremely interesting
compounds with significant anti-HBV activity, and a clear advance
in the art. The data on the anti-HBV effects of .beta.-LddC and
.beta.-L-FddC are summarised in Table 1, below.
Separately, utilizing the above-described procedure, .beta.-L-FddC
was screened for anti-HIV activity. .beta.-L-FddC was tested and
compared to other compounds, and in particular, DDC, .beta.-L-ddC,
alpha-L-FddC, .beta.-L-ddSC and alpha-L-ddSC. The results are
presented in Table 1, below.
Based upon the results set forth in Table 1, .beta.-L-FddC
exhibited anti-HIV activity which was significantly more effective
than ddC, a known anti-HIV agent. The ID.sub.50 concentration of
.beta.-L-FddC required to inhibit viral replication in this assay
was 0.007 micromolar. For ddC, the ID.sub.50 concentration was
determined to be 0.028 micromolar, a 4-fold difference. The
cellular cytotoxicity of .beta.-L-FddC as compared to ddC was also
considerably less as evidenced by the Table 1 data set forth below.
It is interesting to note that this compound has several fold
higher activity against HIV with significantly less cellular
toxicity, an unexpected result. ddC, on the other hand, was more
cytotoxic than .beta.-L-FddC and yet, less active against HIV. In
addition, ddC was shown to exhibit dramatic effects on host
mitochondrial DNA, whereas .beta.-L-FddC had relatively little
effect. It is expected that .beta.-L-FddC would have significantly
lower adverse effects on the host mitochondrial DNA than ddC as
concentrations as high as 100 uM of .beta.-L-FddC were not
inhibitory in the assay. The data on the effects of .beta.-L-FddC
are summarised in Table 1, below and compared with ddC,
.beta.-L-ddC, alpha-L-FddC, .beta.-ddSC and alpha-ddSC. The
implications for .beta.-L-FddC as an anti-HIV agent are clear as
the results presented herein evidence .beta.-L-FddC to be an agent
which exhibits exceptional anti-HIV activity and virtually no
toxicity associated with dose limiting neuropathy. This stands in
contrast to the presently available ddC.
TABLE 1
__________________________________________________________________________
Anti-HBV and Anti-HIV Activities of L-2',3'- Dideoxy Nucleoside
Analogs ID.sub.50 (uM) Cytotoxicity Anti-Mitochondrial Compound CEM
Cells DNA Anti-HBV Anti-HIV
__________________________________________________________________________
ddC 28 0.022 2.8 0.028 .beta.-L--ddC 70 >100 0.01 0.35
.beta.-L--FddC 67 >100 0.01 0.007 alpha-L--FddC >100 ND 0.5
0.3 .beta.-L--ddSC >100 ND >0.5 70 alpha-L--ddSC >100 ND
>0.5 >>100
__________________________________________________________________________
ND -- not determined ddC --
1(2,3-dideoxy-beta-D-ribofuranosyl)cytosine L--ddC --
1(2,3-dideoxy-beta-L-ribofuranosyl)cytosine L--FddC --
1(2,3-dideoxy-beta-L-ribofuranosyl)-5-fluorocytosine alphaL--FddC
-- 1(2,3-dideoxy-alpha-L-ribofuranosyl)-5-fluorocytosine L--ddSC --
1(2,3-dideoxy-4-thio-beta-L-ribofuranosyl)cytosine alphaL--ddSC --
1(2,3-dideoxy-4-thio-alpha-L-ribofuranosyl) cytosine
It is to be understood by those skilled in the art that the
foregoing description and examples are illustrative of practicing
the present invention, but are in no way limiting. Variations of
the detail presented herein may be made without departing from the
spirit and scope of the present invention as defined by the
following claims.
* * * * *